Coenzyme Q10 (CoQ10), commonly known as ubiquinone-10, is present in blood and all body tissues in both reduced and oxidized forms. CoQ10 has an important function in the mitochondrial electron transport chain (ETC) as an electron acceptor and as an electron donor. It is believed that the ETC is comprised of five multi-subunit enzyme complexes, in which CoQ10 and cytochrome c act as shuttles between complex I and III and II and III. Complex I (NADH-ubiquinone oxidoreductase) transfers electron from NADH or NADPH to CoQ10. Complex II (succinate-ubiquinone oxidoreductase) transfers electron from FADH or FMNH to CoQ10. Complex III (ubiquinol-ferrocytochrome c oxidoreductase) transfers electron from reduced CoQ10 (CoQ10H2) to cytochrome c. In addition, CoQ10H2 is one of the antioxidants that protect cells against lipid peroxidation. In the circulation, CoQ10 is mainly carried by lipoproteins, where it is predominantly present in the reduced form. The CoQ10H2 in low density lipoprotein (LDL) is, however, easily oxidized to CoQ10. In fact, CoQ10H2 is the first antioxidant to be depleted when LDL is subjected to oxidative stress in vivo. It has been postulated that CoQ10H2 prevents the initiation and/or the propagation of lipid peroxidation in plasma lipoproteins and biological membranes. The antioxidative activity of CoQ10H2 depends not only on its concentration but also on its redox status. Recent reports have suggested the percentage of CoQ10H2 in total plasma concentration of CoQ10(TQ10) may be lower in patients with atherosclerosis, hyperlipidemia and coronary artery disease, and may be a useful biomarker of oxidative stress. Thus, the measurement of CoQ10H2 and CoQ10 is of primary importance for clinical diagnosis.
Previous CoQ10H2 studies have encountered the problem of CoQ10H2 stability during sample handling, storage, and processing [1, 2, 3, 4, 5, 6, 7]. These studies indicate that CoQ10H2 is unstable in blood, plasma, and hexane extracts at room temperature. Subsequently, the CoQ10H2:TQ10 ratio changes considerably within an hour after the blood sample has been obtained. The lability of CoQ10H2 is due to the hydroquinone moiety which is sensitive to oxygen, and at room temperature, it spontaneously oxidizes to CoQ10 at a rate of ˜2 nM per min. This problem is very obvious in many studies which have reported wide variability in the CoQ10H2:TQ10 ratio in biological fluids [1, 2, 3, 4, 5, 6, 7]. It is believed that sample preparation has a profound effect on the redox status of CoQ10 and that utmost care is required to ensure reliable estimates of the CoQ10H2:TQ10 ratio. Recently, investigators have recommended that each plasma sample be thawed individually, extracted, and analyzed as a continuous process to minimize CoQ10H2 oxidation [8, 9, 10]. This is obviously very impractical for analyzing significant numbers of clinical specimens.
Several in-line post-column reduction methods of CoQ10 to CoQ10H2 have also been reported for simultaneous measurements by electrochemical detection [3, 4]. The complex instrumentation and techniques used in those reports limit their practical value.
In earlier studies, biological fluid samples were converted into either CoQ10 using an oxidizing reagent such as hydrogen peroxide or ferric chloride, or converted into CoQ10H2 using a reducing agent such as sodium tetrahydroborate or sodium dithionite (Table 1). In practice, however, since CoQ10H2 is easily oxidized when exposed to air, CoQ10H2 is susceptible to pre-analytical degradation and analytical error.
Therefore, we developed a simple and rapid procedure using an isocratic HPLC-EC method for simultaneous determination of CoQ10 and CoQ10H2 in human samples. An electrochemical (EC) detector is preferred for detection of CoQ10H2 due to its high sensitivity. The electrochemical reactions are monitored at electrodes that measure the current produced by the reduction of the quinone group of CoQ10 or by the oxidation of the hydroquinone group of CoQ10H2 (FIG. 1). This method may be used to investigate conditions by which the CoQ10H2:TQ10 ratio can be reliably measured.